T-cell contact with antigen-presenting cells (APC) initiates an activation cascade which includes an increase in T-cell intracellular calcium and leads to T-cell proliferation and differentiation. Although T-cell/APC physical contact is required for an immune response, little is known about the patterns of cellular interaction and their relation to activation. We have combined fluorescence spectroscopy and imaging with optical manipulation to investigate the physical properties of T-cell activation. We study cell-cell contact requirements for T-cell activation using optical tweezers to control the orientation of T-cell/APC pairs and fluorescence microscopy to measure the subsequent T-cell intracellular calcium level ([Ca2+]i) response. T cells which are presented with antigen at the leading edge have a higher probability of responding and a shorter latency of response than those contacting APCs or antibody-coated beads with their trailing end. Alterations in anti body dens ity and bead size are used to determine the spatial requirements for T cell activation and the minimum number of receptors which must be engaged in order to transmit a positive signal. Results show that T cell responses (response percentage, latency and [Ca2+]i pattern) depend on both antibody density on bead and bead size. ~340 TCRs are required to be engaged for intracellular calcium level increase in T cell activation.